Modulation of flagellum attachment zone protein FLAM3 and regulation of the cell shape in Trypanosoma brucei life cycle transitions

The cell shape of Trypanosoma brucei is influenced by flagellum-to-cell-body attachment through a specialised structure – the flagellum attachment zone (FAZ). T. brucei exhibits numerous morphological forms during its life cycle and, at each stage, the FAZ length varies. We have analysed FLAM3, a large protein that localises to the FAZ region within the old and new flagellum. Ablation of FLAM3 expression causes a reduction in FAZ length; however, this has remarkably different consequences in the tsetse procyclic form versus the mammalian bloodstream form. In procyclic form cells FLAM3 RNAi results in the transition to an epimastigote-like shape, whereas in bloodstream form cells a severe cytokinesis defect associated with flagellum detachment is observed. Moreover, we demonstrate that the amount of FLAM3 and its localisation is dependent on ClpGM6 expression and vice versa. This evidence demonstrates that FAZ is a key regulator of trypanosome shape, with experimental perturbations being life cycle form dependent. An evolutionary cell biology explanation suggests that these differences are a reflection of the division process, the cytoskeleton and intrinsic structural plasticity of particular life cycle forms

Lineage-specific proteins essential for endocytosis in trypanosomes

Clathrin-mediated endocytosis (CME) is the most evolutionarily ancient endocytic mechanism known, and in many lineages the sole mechanism for internalisation. Significantly, in mammalian cells CME is responsible for the vast bulk of endocytic flux and has likely undergone multiple adaptations to accommodate specific requirements by individual species. In African trypanosomes, we previously demonstrated that CME is independent of the AP-2 adaptor protein complex, that orthologues to many of the animal and fungal CME protein cohort are absent, and that a novel, trypanosome-restricted protein cohort interacts with clathrin and drives CME. Here, we used a novel cryomilling affinity isolation strategy to preserve transient low-affinity interactions, giving the most comprehensive trypanosome clathrin interactome to date. We identified the trypanosome AP-1 complex, Trypanosoma brucei (Tb)EpsinR, several endosomal SNAREs plus orthologues of SMAP and the AP-2 associated kinase AAK1 as interacting with clathrin. Novel lineage-specific proteins were identified, which we designate TbCAP80 and TbCAP141. Their depletion produced extensive defects in endocytosis and endomembrane system organisation, revealing a novel molecular pathway subtending an early-branching and highly divergent form of CME, which is conserved and likely functionally important across the kinetoplastid parasites

Regulation of Toll-like receptor signaling by NDP52-mediated selective autophagy is normally inactivated by A20

Toll-like receptor (TLR) signaling is linked to autophagy that facilitates elimination of intracellular pathogens. However, it is largely unknown whether autophagy controls TLR signaling. Here, we report that poly(I:C) stimulation induces selective autophagic degradation of the TLR adaptor molecule TRIF and the signaling molecule TRAF6, which is revealed by gene silencing of the ubiquitin-editing enzyme A20. This type of autophagy induced formation of autophagosomes and could be suppressed by an autophagy inhibitor and lysosomal inhibitors. However, this autophagy was not associated with canonical autophagic processes, including involvement of Beclin-1 and conversion of LC3-I to LC3-II. Through screening of TRIF-interacting ‘autophagy receptors’ in human cells, we identified that NDP52 mediated the selective autophagic degradation of TRIF and TRAF6 but not TRAF3. NDP52 was polyubiquitinated by TRAF6 and was involved in aggregation of TRAF6, which may result in the selective degradation. Intriguingly, only under the condition of A20 silencing, NDP52 could effectively suppress poly(I:C)-induced proinflammatory gene expression. Thus, this study clarifies a selective autophagic mechanism mediated by NDP52 that works downstream of TRIF–TRAF6. Furthermore, although A20 is known as a signaling fine-tuner to prevent excess TLR signaling, it paradoxically downregulates the fine-tuning effect of NDP52 on TLR signaling

A Myo6 Mutation Destroys Coordination between the Myosin Heads, Revealing New Functions of Myosin VI in the Stereocilia of Mammalian Inner Ear Hair Cells

Myosin VI, found in organisms from Caenorhabditis elegans to humans, is essential for auditory and vestibular function in mammals, since genetic mutations lead to hearing impairment and vestibular dysfunction in both humans and mice. Here, we show that a missense mutation in this molecular motor in an ENU-generated mouse model, Tailchaser, disrupts myosin VI function. Structural changes in the Tailchaser hair bundles include mislocalization of the kinocilia and branching of stereocilia. Transfection of GFP-labeled myosin VI into epithelial cells and delivery of endocytic vesicles to the early endosome revealed that the mutant phenotype displays disrupted motor function. The actin-activated ATPase rates measured for the D179Y mutation are decreased, and indicate loss of coordination of the myosin VI heads or ‘gating’ in the dimer form. Proper coordination is required for walking processively along, or anchoring to, actin filaments, and is apparently destroyed by the proximity of the mutation to the nucleotide-binding pocket. This loss of myosin VI function may not allow myosin VI to transport its cargoes appropriately at the base and within the stereocilia, or to anchor the membrane of stereocilia to actin filaments via its cargos, both of which lead to structural changes in the stereocilia of myosin VI–impaired hair cells, and ultimately leading to deafness

Form, fabric, and function of a flagellum-associated cytoskeletal structure.

Trypanosoma brucei is a uniflagellated protist and the causative agent of African trypanosomiasis, a neglected tropical disease. The single flagellum of T. brucei is essential to a number of cellular processes such as motility, and has been a longstanding focus of scientific enquiry. A number of cytoskeletal structures are associated with the flagellum in T. brucei, and one such structure—a multiprotein complex containing the repeat motif protein TbMORN1—is the focus of this review. The TbMORN1-containing complex, which was discovered less than ten years ago, is essential for the viability of the mammalian-infective form of T. brucei. The complex has an unusual asymmetric morphology, and is coiled around the flagellum to form a hook shape. Proteomic analysis using the proximity-dependent biotin identification (BioID) technique has elucidated a number of its components. Recent work has uncovered a role for TbMORN1 in facilitating protein entry into the cell, thus providing a link between the cytoskeleton and the endomembrane system. This review summarises the extant data on the complex, highlights the outstanding questions for future enquiry, and provides speculation as to its possible role in a size-exclusion mechanism for regulating protein entry. The review additionally clarifies the nomenclature associated with this topic, and proposes the adoption of the term “hook complex” to replace the former name “bilobe” to describe the complex

The bilobe structure of Trypanosoma brucei contains a MORN-repeat protein

10.1016/j.molbiopara.2009.05.001Molecular and Biochemical Parasitology167295-103MBIP

Unexpected plasiticty in the life cycle of Trypanosoma brucei

African trypanosomes cause sleeping sickness in humans and nagana in cattle. These unicellular parasites are transmitted by the bloodsucking tsetse fly. In the mammalian host’s circulation, proliferating slender stage cells differentiate into cell cycle-arrested stumpy stage cells when they reach high population densities. This stage transition is thought to fulfil two main functions: first, it auto-regulates the parasite load in the host; second, the stumpy stage is regarded as the only stage capable of successful vector transmission. Here, we show that proliferating slender stage trypanosomes express the mRNA and protein of a known stumpy stage marker, complete the complex life cycle in the fly as successfully as the stumpy stage, and require only a single parasite for productive infection. These findings suggest a reassessment of the traditional view of the trypanosome life cycle. They may also provide a solution to a long-lasting paradox, namely the successful transmission of parasites in chronic infections, despite low parasitemia